Recently, the use of surface expression to produce valuable exogenous proteins on cell surfaces has been attempted with bacteriophages, bacteria, and yeast for the purpose of creating new vaccines, screening various kinds of antigens and antibodies, and fixing useful enzymes onto cell surfaces.
Originally, the idea of expressing exogenous proteins on a cell surface was to produce antigenic regions of peptides, especially for the large-scale stable expression of vaccines. Currently, pathogenic bacteria are randomly mutated to produce vaccines and screened to collect bacteria with consistent and stable titers. However, unfortunately, the enzymatic activity is invariably lost after oral administration to humans and animals. Therefore, many studies have been conducted to over come this problem. Normally, the cell surface protein of a Gram-negative bacterium is adopted and its gene ligated with an antigenic protein gene, which is then introduced to proper host cells so that fusion proteins are efficiently produced on the cell surface. The recombinant protein prepared through this procedure can be an effective antigen as it is protruded onto the cell surface. In particular, Gram-negative bacteria have been reported as most suitable for production, as the lipopolysaccharides (LPS) in the cell outer membrane enhance the antigenicity of the proteins expressed on the cell surface.
To express exogenous proteins on a cell surface, the presence of a secretion signal is required within the primary sequence, since this passes the biosynthesized cell proteins through the cell membrane. Besides, in Gram-negative bacteria, the recombinant protein must also pass though the cell inner membrane and space between the cell membranes, be inserted and attached to the cell outer membrane, and finally stably protruded to the external side of the cell membrane.
Practically, there are certain proteins that include such a secretion signal and targeting signal and are stably protruded onto the cell surface, for example, cell surface proteins, specific enzymes, and toxin proteins. As such, if these secretion and targeting signals are associated with a proper promoter, exogenous proteins can be successfully expressed onto a bacterial surface.
In general, the cell surface proteins adopted for the surface expression of foreign proteins can be basically classified to 4 kinds, a cell outer membrane, lipoprotein, secretion protein, and cell surface organ protein. Until now, the surface proteins present in Gram-negative bacteria, for example, LamB, PhoE, and OmpA, have been mainly utilized to produce useful foreign proteins. However, these proteins present structural restrictions as regards the size of the insertable proteins, which are inserted into the protruded loop on the cell surface. Since the C- and N-termini of the inserted exogenous protein should be stereochemically close, if they are distant, connected peptides can be ligated to reduce the distance between the two termini.
Concretely, if LamB and PhoE are used to insert an exogenous polypeptide consisting of more than 50˜60 amino acids, structural constraints are invoked preventing the creation of a stable protein on the cell membrane (Charbit, et al., J. Immunol., 139: 1658-1664, 1987; Agterberg, et al., Vaccine, 8: 85-91, 1990). Although OmpA can be utilized to introduce exogenous proteins into the protruded loop, only a partial fragment of OmpA containing a minimal targeting signal can actually be added due to the structural constraint. β-lactamase has been expressed on a cell surface by connecting the OmpA targeting signal at the C-terminus.
Recently, the ice-nucleation protein (INP) derived from Pseudomonas sp. was found to be a cell outer membrane of Gram-negative bacteria and utilized for surface expression (Jung et al., Nat. Biotechnol., 16: 576-580, 1998; Jung et al., Enzyme Microb. Technol., 22(5): 348-354, 1998; Lee et al., Nat. Biotechnol., 18: 645-648, 2000). Jung and colleagues expressed levansucrase onto a cell surface using the ice nucleation protein, consisting of the N-terminus, central repetitive region, and C-terminus, and ligating the levansucrase gene at the C-terminus, while also expressing carboxymethylcellulase using the ice nucleation protein, consisting of the N-terminus, deleted central repetitive region, and C-terminus, and fusing the gene at the C-terminus, so as to assay the respective enzymatic activities. In addition, Lee and colleagues used the ice-nucleation protein, comprising of just the N-terminus or the N-terminus and C-terminus, ligated with the hepatitis B virus surface antigen and hepatitis C virus core antigen at each terminus, for expression on the cell surface of an Escherichia coli or Salmonella typhi Ty21a strain, then confirmed that these proteins were effective for complex live vaccines.
Lipoproteins have also been utilized as a surface protein for surface expression. In particular, E. coli lipoproteins can pass through the cell inner membrane based on the secretion signal at the N-terminus and contain L-cystein at the terminus directly connected to the cell outer membrane or inner membrane. A major lipoprotein, Lpp, is associated with the cell outer membrane at the N-terminus and with peptidoglycan (PG) at the C-terminus. Thus, if Lpp is connected with the OmpA fragment of the cell outer membrane protein, exogenous proteins can be stably expressed onto the cell surface of the cell outer membrane (Francisco, et al., Proc. Natl. Acad. Sci. USA, 89: 2713-2717, 1992). This characteristic has also been used with another lipoprotein, TraT, to express foreign peptides, such as the C3 epitope of the poliovirus, onto a cell surface (Felici, et al., J. Mol. Biol., 222: 301-310, 1991). Furthermore, the peptidoglycan-associated lipoprotein (PAL), although not yet elucidated as regards its precise function, has been adopted to produce recombinant antigens through surface expression (Fuchs, et al., Bio/Technology, 9: 1369-1372, 1991). In this case, the C-terminus of PAL is ligated to the cell wall and the N-terminus to the recombinant antibody so as to express a fusion protein on the cell surface.
Meanwhile, even though secretion proteins that can pass through the cell outer membrane can be used as a surface protein, this has not been developed in Gram-negative bacteria and only a few kinds of secretion proteins can help passage through the cell outer membrane in the presence of specific proteins participating in the secretion mechanism. For example, Klebsiella sp. pullulanase as a lipoprotein is completely secreted into a cell culture medium after its N-terminus is substituted with a lipid substance and attached to the cell outer membrane. Kornacker and colleagues expressed β-lactamase onto a cell surface when using the N-terminus fragment of pullulanase, yet the resulting fusion protein of pullulanase-β-lactamase was instantly attached onto the cell surface, then unfortunately separated into the cell culture medium. In addition, this process has also been exploited to produce alkaline phosphatase, a periplasmic space protein, yet the recombinant protein is not stably expressed as at least 14 proteins are required for the secretion (Kornacker, et al., Mol. Microbiol., 4: 1101-1109, 1990).
Moreover, IgA protease, derived from the pathogenic microbe Neisseria sp., has a specific secretion system with a fragment signal present at the C-terminus, which makes the protease present at the N-terminus stably attached to the cell outer membrane. Once arriving at the cell outer membrane and protruding on the cell surface, the protease is secreted into the cell culture medium based on its hydrolytic capacity. Klauser and colleagues inconsistently expressed the B subunit of the cholera toxin with a molecular weight of about 12 kDa onto a cell surface using this IgA protease fragment (Klauser, et al., EMBO J., 9: 1991-1999, 1990). However, the secretion of the fused protein was inhibited by the protein folding induced in the cell membrane space during the secretion process.
Besides, in the case of Gram-negative bacteria, the cell suborgans present on the cell surface and applicable for surface expression are composed of flagella, pili, and fimbriae etc. In detail, the B subunit of the cholera toxin and peptides derived from the hepatitis B virus have been consistently produced using flagellin as a subunit composed of flagella and identified as strongly binding with their antibodies (Newton, et al., Science, 244:70-72, 1989). Then, fimbrin, a subunit constituting of threadlike fimbriae on the cell surface, has been utilized to express exogenous peptides, yet only small peptides have been successfully produced (Hedegaard, et al., Gene, 85: 115-124, 1989).
Although the surface proteins of Gram-negative bacteria have already been used to perform surface expression, recently, the surface proteins of Gram-positive bacteria have also been used for surface expression (Samuelson, et al., J. Bacteriol., 177: 1470-1476, 1995). Yet, even in this case, a secretion signal for passing through the cell inner membrane and carrier for surface expression and attaching onto the cell membrane are also needed. In fact, the secretion signal of the lipase derived from Staphylococcus hyicus and membrane attachment carrier of protein A derived from Staphylococcus aureus have been utilized to produce a malaria blood stage antigen composed of 80 amino acids and albumin attachment protein derived from Streptococcus protein G and efficiently express the resulting proteins onto the cell surface.
As described above, since much research has already focused on surface expression with Gram-negative bacteria and Gram-positive bacteria, a number of expression systems have already been developed for the production of valuable proteins and submitted for patent applications, especially in the USA, Europe, and Japan. In detail, 5 patent cases have disclosed the use of the cell outer membrane proteins of Gram negative bacteria (WO 9504069, WO 9324636, WO 9310214, EP 603672, U.S. Pat. No. 5356797), one patent application has reported the use of pili as a cell surface organelle (WO 9410330), and one case using a cell surface lipoprotein (WO 9504079).
As stated above, to express exogenous proteins onto a cell surface using a cell outer membrane protein, the proper cell inner membrane and exogenous protein must be connected on a gene level, induced for biosynthesis, and sustained on the cell outer membrane after passing stably through the cell inner membrane. To accomplish this procedure, a cell inner membrane satisfying the following requirements should be selected, then applied to the carrier for surface expression: above all, the presence of a secretion signal for passing through the cell inner membrane, second, the presence of a targeting signal for stable attachment to the cell outer membrane, third, massive expression onto the cell surface, and fourth, stable expression of the protein, regardless of its size.
However, carriers for surface expression that meet all these requirements have not yet been developed. Currently, only the following disadvantages have been remedied.
Based on such a background, the present inventors investigated the application of a poly-γ-glutamate synthase gene (pgsBCA) derived from a Bacillus sp. strain as a novel carrier for surface expression. As a result, a novel expression vent or pgsBCA-containing gene that can efficiently produce exogenous proteins onto microbial surfaces was developed along with a method for successfully expressing exogenous proteins onto microbial surfaces on a large scale.